This proposal presents a program of investigation whereby purification and characterization of the proteolytic enzymes found in Pronase will be continued. The research will include the purification and characterization of the higher-molecular-weight components; this would include an analysis of the possibility that some of these components may act as precursors for the already well-characterized lower-molecular-weight proteases. Other studies will include the further characterization of an aminopeptidase emphasizing the structure-function relation of the protein through chemical modification studies and also the beginning characterization of its primary structure. Studies will also be initiated towards the application of glass-bound aminopeptidase as a tool in the sequence analysis of polypeptide chains. Sequence studies will be carried out on Pronase carboxypeptidase to confirm the preliminary data that this enzyme is homologous with bovine carboxypeptidase. Other studies will be directed towards the unique susceptibility to apparent denaturation after acetylation of a very stable chymoelastase. Studies will be directed towards elucidating those structures which are affected by acetylation in the absence of glycerol where marked denaturation occurs. A further area which we wish to explore is the preparation of glass-bound stable Pronase endopeptidases and exopeptidases together with an immobilized prolidase so as to be able to digest enzymatically substrate polypeptide chains and thereby gain more precise amino acid compositions. Because four of the proteases have been discovered to be both stable and active in high concentrations of denaturant, studies will be carried out to determine whether the specificities of these enzymes are the same in the presence and absence of denaturant.